Classification of tissue cultures based

on the origin of the cells

• Primary culture (directly from animal or plant tissue)
• Extended culture (multipassage culture) – cell strain
• Established (transformed) cell lines

Primary culture

Survival times of primary neuronal

Primary tissue culture cells in culture
• A culture derived directly from a tissue
– A stage from cell isolation to first subculturing
– Carrot callus growth was primary plant culture
• Best resembling natural tissue
• Limited growth potential
• Limited life span
• May give rise to a cell strain
or be immortalized
• Strain – a lineage of cells
originating from one primary
culture

Steps in primary tissue culture Isolation of tissue

• Isolation of tissue • Make sure your work is within rules
• Disaggregation of cells – initiation of culture • Work safely, especially with human tissue
• Incubation and growth • If you isolate your cells far from culture place (as it is in
our case) keep it on ice (4oC) for up to 72 hours

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 Disaggregation of cells Explant culture
• Cells can be allowed to migrate out from an explant
• Mechanical dissociation (mincing)
• Enzymatic dissociation

Exception – hematopoietic cells do not need to be

disaggregated, they already are

Explant culture Enzymatic disaggregation

• Involves placing a piece of tissue into the tissue culture • Avoids selection of cells by migration and usually yields
dish and allowing cells to migrate out from the tissue more representative sample
• First type of cell culture developed – But still selects by resistance to enzymatic treatment
• Performed in the case of cells which are protease sensitive • Faster than explant
– Smooth muscle cells, bone cells
• Or in case of small amount of tissue (such as needle
biopsies)
• Not very effective for cells with poor adhesion (migration)
• Fibrinogen and thrombin used to stimulate adhesion
• Disadvantages – selection by speed of migration, type of
attachment, localization within tissue etc.

Enzymatic disaggregation Physical connections between cells

• Cell to cell adhesion is mediated by a variety of cell • Cells in multicellular organisms are in contact with each
adhesion molecules other or extracellular matrix
• The connections between cells and extracellular matrix • Cell connections involve multiple ligands and cell adhesion
have to be broken receptors
• To break calcium dependent adhesion (cadherins and • The interaction between cell
selectins) we use EDTA or EGTA (both calcium chelators) adhesion receptors and their
• Extracellular matrix proteins such as fibronectin and ligands are relatively weak
laminin are protease sensitive – A lot of weak interactions
• Proteoglycans can be partially degraded by hyaluronidase make a strong bond
or heparinase

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 Principal classes of cell-adhesion
receptors Extracellular matrix
• Cadherins • Network of proteins and carbohydrates that binds cells
• Ig-family of cell adhesion molecules together
• Integrins – Supports and surrounds cells
• Selectins – Regulates cells activities
• Others such as • Only 5 classes of macromolecules
– Mucins – Collagens
– Connexins – Elastic fibers
– Proteoglycans
– Hyaluronan
– Adhesive glycoproteins
• They can be mixed up in different proportions for different
functions

Enzymes used in enzymatic disaggregation More rules

• Enzymes • Start with trypsin/EDTA and than proceed to more
– Trypsin complex enzymes
– Collagenase II (from Pseudomonas perfringens) • Warm or cold trypsin
– Elastase • Cold seems to give a higher yields but warm is faster
– Hyaluronidase (shorter exposure)
– DNase • If using warm trypsin collect cells every half hour to avoid
cell death from exposure (remember to inactivate and
– Pronase (bacterial protease) remove trypsin before plating cells)
• Usually a combination of enzymes • Warm trypsin works better with big amounts of young
• Crude preparations are usually more efficient tissue (mouse or chick embryos) and not too well with
– The purer the less toxic adult tissue (more connective tissue)
– The cruder the more effective due to contamination
with other proteases

Cold trypsin Other enzymatic procedures

• Cold trypsin – avoids damage by exposure to warm trypsin
• Allows for enzyme penetration with minimum of enzymatic
activity
• Followed by faster 37oC digestion time
• Gives higher yield and higher survival rate
• Preserves more different cell types
• Convenient
• Some tissues such as fibrous connective tissue are
resistant to trypsin
• Collagenase – particularly connective tissue and muscle
• Hyaluronidase – to dissolve proteoglycans
• Pronase and dispase – bacterial proteases
• DNase – to dissolve DNA aggregates from damaged cells

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 Mechanical disaggregation Incubation and growth
• Produces cell suspension quicker than other methods • Appropriate medium supplemented with growth factors,
• But causes more mechanical damage cytokines and all the goodies
• Several methods • Some cells require special adhesion surfaces (cover tissue
– Mincing culture dish with extracellular matrix proteins or synthetic
attachment molecules)
– Collecting cells when tissue is sliced
• Transfer cells to final growing conditions as soon as
– Pressing the tissue through a series of sieves possible
– Repeated pipetting • Challenges
– Removal of dead cells
– Enrichment of viable cells
– Separation of cell types

Separation of nonviable cells Separation of cell types

• For adherent cultures first change of media • Selective media
• Gradual dilution of suspension cells when proliferation • Difference in the speed of attachment
starts • Use of enzymes
– Collagenase does not easily disperse epithelial cells but
works well on stroma
• Neurons need NGF while glial cells don’t

Adrenal Glands Histology of adrenal gland

• Also called the suprarenal glands • Produce glucocorticoids, mineralocorticoids, steroids,
• Located above the kidneys (cortex) and catecholamines (medulla)
• Highest blood flow rate in the body
• Two Regions
– Medulla (inner)
– Cortex (outer)
• Medulla & cortex are functionally different glands
– Embryological origins:
• Medulla derives from ectoderm (neural crest)
• Cortex derives from mesoderm
– Is actually two separate organs in amphibians

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 Function of adrenal medulla
• Pheochromocytes also called chromaffin cells are modified,
axonless neurons with a purely secretory function
– Chromaffin cells because they stain dark with chromium
• Chromaffin cells produce primarily epinephrine
(adrenaline), can also produce norepinephrine, but not
both
– Essential for fight or flight response
• Other hormones produced by the medulla are: dopamine,
met-enkephalin (opioids), ADH, NPY, and adrenomedullin
Basic rules
• Remove fat and dead tissue
• Use sharp instruments for dissection to avoid cell damage
• Do not leave enzymes in the cell suspension
• Use high concentration of cells when seeding (much
higher than for cell lines)
• Use appropriate, rich media
• Remember that primary cells might require very specific
growth factors or attachment factors
• Embryonic tissue gives better results

Cold trypsin – the procedure

• Sterilize
• Remove fat and dead tissue
• Slice adrenal gland in half
• Cut out a piece of the medulla
• Wash it twice
• Add cold trypsin
• Cold incubation
• Remove trypsin
• Warm incubation
• Plating